Project Summary Methionine occupies a special place among amino acids. This is best illustrated by the phenomenon called ?methionine-dependence of cancer?. This cancer specific metabolic need describes the behavior of cells when grown in medium lacking methionine but supplemented with the immediate metabolic precursor homocysteine. Non-tumorigenic cells maintain their proliferation rate in homocysteine, but the vast majority of cancer cells, independent of their tissue origin, induce cell cycle arrest followed by apoptosis when cultured in homocysteine medium. Importantly, methionine- dependence is not only observed in cultured cancer cells. Solid tumors and leukemias also depend on high flux through the metabolic pathways connected to methionine. Furthermore, longevity is strikingly connected to dietary methionine uptake. Caloric restriction is well known to increase longevity in many organisms. This effect is mimicked by restricting methionine in an otherwise rich diet. Conversely, supplementing a low-calorie diet with methionine eliminates the benefits of caloric restriction for longevity. This proposal seeks understanding of the molecular effects that fluctuating methionine levels have on cellular and organismal physiology, as well as an explanation for the methionine dependence of cancer. Reports in the literature and our preliminary studies suggest that methionine uses unique signaling pathways that have not been explored at the molecular level. We find that the canonical amino acid and nutrient responsive TOR pathway is not involved in measuring or signaling methionine levels. Furthermore, the downstream metabolites S-adenosylmethionine (SAM) and S-adenosyl- homocysteine (SAH) ? and not methionine itself ? appear to be the effector metabolites for both the effects on cancer cell proliferation and longevity. SAM is the primary cellular methyl donor and the SAM/SAH ratio is generally considered the determinant of the cellular methylation potential. As such these metabolites are ideally positioned to signal methionine levels through specific methylation events. We have identified methylation events on groups of RNAs and specific proteins as candidates that link methionine levels to specific cellular responses. One goal of this proposal is to identify the critical RNAs and proteins that are controlled through methylation and show a hypersensitive response to fluctuations in methionine or SAM/SAH concentrations. The sensitive reaction to varying methylation allows these RNAs and proteins to trigger signals and ultimately cellular pathways that connect methionine metabolism to cell proliferation and other cellular functions. The second goal of the proposal is thus to identify these pathways and initiate investigation of how they connect metabolism with cell physiology at the molecular level. Understanding the molecular concepts that integrate methionine metabolism with other cellular functions promise new therapeutic strategies for treatment of cancer and other age-related disorders. Thus, this proposal aims to development molecular insight into a fundamental, so far molecularly unexplored, biological process with great potential for therapeutic exploitation.